Identification of substrates and regulators of the mitogen-activated protein kinase ERK5 using chimeric protein kinases.
Extracellular signal-regulated protein kinase 5 (ERK5) is a recently discovered orphan mitogen-activated protein kinase for which no substrates or strong activators have been described. Two ERK5 chimeras were created as a novel approach to discover its substrates and upstream regulators. One chimeric protein contained the N-terminal domain of the ERK5 catalytic core (subdomains I-IV) and the C-terminal domain of the ERK2 catalytic core (subdomains V-XI). This chimera was highly responsive to stimuli that regulate ERK2 in vitro and in cells. A second chimeric protein consisted of the N-terminal domain of ERK2 (subdomains I-IV) and the C-terminal domain of the ERK5 catalytic core (subdomains V-XI). This chimera was activated in bacteria by coexpression with a constitutively active mutant of MEK1. Using the activated chimera, we identified three in vitro substrates of ERK5. Assaying ERK5 activity in immune complexes with one of these substrates, c-Myc, we determined that the ERK5 catalytic domain is activated by V12 H-Ras and to a lesser extent by phorbol ester but not by constitutively active mutants of Raf-1. Thus, ERK5 is a target of a novel Ras effector pathway that may communicate with c-Myc.
English
J M
JM
Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
Pearson
G
G
Baer
R
R
Cobb
M H
MH
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D048054:Mitogen-Activated Protein Kinase 7; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018631:ras Proteins / Q000378:metabolism
D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048054:Mitogen-Activated Protein Kinase 7; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate